DK149091B - METHOD OF ANALOGUE FOR THE PREPARATION OF PEPTIDS CONTAINING 2-7 AMINO ACID RESIDUES, DESAMINODIPEPTIDES OR AMIDES THEREOF WITH UBIQUITIN SIMILAR ACTIVITY OR PHARMACEUTICAL ACCEPTABLE ACID ADDITIONAL SALTS - Google Patents

METHOD OF ANALOGUE FOR THE PREPARATION OF PEPTIDS CONTAINING 2-7 AMINO ACID RESIDUES, DESAMINODIPEPTIDES OR AMIDES THEREOF WITH UBIQUITIN SIMILAR ACTIVITY OR PHARMACEUTICAL ACCEPTABLE ACID ADDITIONAL SALTS Download PDF

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DK149091B
DK149091B DK109180AA DK109180A DK149091B DK 149091 B DK149091 B DK 149091B DK 109180A A DK109180A A DK 109180AA DK 109180 A DK109180 A DK 109180A DK 149091 B DK149091 B DK 149091B
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Gideon Goldstein
George Heavner
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Abstract

Peptides of the following formula:wherein A is a member selected from the group consisting of deamino-GLN, GLN, and (SAR)<sub>n</sub>-GLN; n is an integer from to 4; and B is a member selected from the group consisting of LYS-OH, LYS-NH<sub>2</sub>, decarboxy-LYS,LYS-SAR-OH and LYS-SAR-NH<sub>2</sub>, have significantly increased ubiquitin-like activity. Also provided are therapeutic compositions and methods for use thereof.

Description

149091149091

Den foreliggende opfindelse angår en analogifremgangsmåde til fremstilling af hidtil ukendte peptider indeholdende 2-7 aminosyrere-ster, desaminodipeptider eller amider deraf med ubiquitin-lignende aktivitet eller farmaceutisk acceptable syreadditionssalte deraf.The present invention relates to an analogous method for the preparation of novel peptides containing 2-7 amino acid residues, desaminodipeptides or amides thereof with ubiquitin-like activity or pharmaceutically acceptable acid addition salts thereof.

05 USA-patentskrift nr. 4.002.602 med benævnelsen "Ubiquitous05 US Patent No. 4,002,602 entitled "Ubiquitous

Immunopoietic Polypeptide and Methods11, hvori den ene af de foreliggende opfindere er opfinder, omhandler det langkædede polypep-tid, ubiquitin (der kaldes "Ubiquitous Immunopoietic Polypeptide).Immunopoietic Polypeptide and Methods11, in which one of the present inventors is an inventor, relates to the long-chain polypeptide, ubiquitin (called "Ubiquitous Immunopoietic Polypeptide).

BE patentskrift nr. 864.122 omhandler pentapeptidet med sekvensen 10 H-X-Y-Z-GLN-LYS-OH, hvori X betegner TYR eller ALA, Y betegner ASN eller ALA og Z betegner ILE eller ALA. Denne patentansøgning beskriver, at dette pentapeptid har lignende biologisk aktivitet som det langkædede polypeptid, kendt som ubiquitin. Pentapeptidet med formlen H-TYR-ASN-ILE-GLN-LYS-OH (der undertiden her 15 omtales som "ubiquitin-pentapeptid"), som var den mest aktive blandt de i nævnte ansøgning beskrevne forbindelser, udviste aktivitet ved muse-testen i eksempel II deraf ved koncentrationer varierende fra 10 ng/ml til 1 pg/ml. Det var også beskrevet, at pentapeptiderne med formlen H-ALA-ASN-ILE-GLN-LYS-OH, H-TYR-ALA-ILE-GLN-20 LYS-OH og H-TYR-ASN-ALA-GLN-LYS-OH besad aktivitet ved kyl-linge-testen i eksempel XIV til XVI deri ved en koncentration på 0,1 pg/ml (100 ng/ml).BE patent specification 864,122 discloses the pentapeptide having the sequence 10 H-X-Y-Z-GLN-LYS-OH, wherein X represents TYR or ALA, Y represents ASN or ALA and Z represents ILE or ALA. This patent application discloses that this pentapeptide has similar biological activity to the long chain polypeptide known as ubiquitin. The pentapeptide of formula H-TYR-ASN-ILE-GLN-LYS-OH (sometimes referred to herein as "ubiquitin pentapeptide"), which was the most active of the compounds described in said application, exhibited activity in the mouse test in Example II thereof at concentrations ranging from 10 ng / ml to 1 pg / ml. It was also disclosed that the pentapeptides of formula H-ALA-ASN-ILE-GLN-LYS-OH, H-TYR-ALA-ILE-GLN-20 LYS-OH and H-TYR-ASN-ALA-GLN-LYS-OH OH exhibited activity in the chickling test of Examples XIV to XVI therein at a concentration of 0.1 µg / ml (100 ng / ml).

Der henvises til ovennævnte patentskrifter med hensyn til en omtale af anden kendt teknik og de i forbindelse med den foreliggen-25 dø opfindelse involverede biologiske processer.Reference is made to the above-mentioned patents for reference to other prior art and to the biological processes involved in the present invention.

Den foreliggende opfindelse tilvejebringer peptider med en fra de ovennævnte kendte peptider væsentligt forskellig aminosyrese-kvens, og det er derfor i sig selv overraskende, at de omhandlede forbindelser besidder ubiquitin-lignende aktivitet. Hertil kommer, at 50 de omhandlede forbindelser er mindst lige si aktive som de nævnte kendte pentapeptider, mens de for det meste har væsentligt simplere struktur. Disse peptider indebærer derfor også en væsentlig fordel med hensyn til fremstillingslethed og omkostninger. Nogle af disse hidtil ukendte peptider er også signifikant mere aktive end de nævn-35 te kendte pentapeptider.The present invention provides peptides with a substantially different amino acid sequence from the above known peptides, and it is therefore surprising in itself that the compounds of this invention possess ubiquitin-like activity. In addition, the compounds of the present invention are at least as active as the aforementioned known pentapeptides, while for the most part they have substantially simpler structure. Therefore, these peptides also have a significant advantage in terms of ease of production and cost. Some of these novel peptides are also significantly more active than the aforementioned known pentapeptides.

Den foreliggende opfindelse tilvejebringer således en analogifremgangsmåde til fremstilling af hidtil ukendte peptider med den i indled- 149091 2 ningen til krav 1 angivne formel eller farmaceutisk acceptable syreadditionssalte deraf, som har ubiquitin-lignende aktivitet ved at de inducerer differentiering af både T-prækursor-celler og B-prækursor-celler til hhv. T-celler og B-celler, og som derfor er særligt værdi-05 fulde i immunsystemet hos mennesker og dyr.Thus, the present invention provides an analogous method for preparing novel peptides of the formula or pharmaceutically acceptable acid addition salts thereof set forth in the preamble of claim 1 which have ubiquitin-like activity in inducing differentiation of both T precursor cells. and B precursor cells for, respectively. T-cells and B-cells, which are therefore particularly valuable-05 in the human and animal immune system.

De omhandlede hidtil ukendte peptider har den almene formel:The novel peptides of this invention have the general formula:

A-BA-B

10 hvori A betegner en gruppe udvalgt blandt desamino-GLN, GLN og (SAR)n-GLN, hvor n betegner et helt tal fra 1 til 4, og B betegner en gruppe udvalgt blandt LYS-OH, LYS-NHg og LYS-SAR-NHg. Forbindelserne, hvori A er desamino-GLN, og B er LYS-OH eller LYS-NH2, er desaminodipeptider, men kaldes i nærværende sammen-15 hæng for peptider.Wherein A represents a group selected from desamino-GLN, GLN and (SAR) n-GLN, where n represents an integer from 1 to 4, and B represents a group selected from LYS-OH, LYS-NHg and LYS-SAR. -NHg. The compounds in which A is desamino-GLN and B is LYS-OH or LYS-NH2 are desaminodipeptides, but are herein referred to as peptides.

Fremgangsmåden ifølge opfindelsen er ejendommelig ved, at man i) binder en sarcosin eller Ne-beskyttet lysin, der er beskyttet på α-aminogruppen, til en polymer-harpiks ved covalent binding, hvilken polymer er en sådan, som indeholder en 20 funktionel gruppe, hvortil den første beskyttede aminosyre kan bindes fast ved hjælp af den covalente binding, ii) fjerner den a-amino-beskyttende gruppe fra aminosyrehar- a £ piksen, og om nødvendigt binder lysin, der er N - og N -beskyttet, til SAR-harpiksen og fjerner a-aminobeskyttel-25 sesgruppen fra LYS-SAR-harpiksen, iii) kobler glutamin eller desaminoglutamin, som er beskyttet pi dens eventuelle α-aminogruppe og pi dens ε-amino-gruppe, til B-aminosyre-harpiksen, og iv) når A-gruppen indeholder en eller flere sarcosyl-dele, 30 fjerner a-aminobeskyttelsesgruppen fra GLN-B-harpiksen og binder sarcosin, som er beskyttet på dens a-aminogruppe, til kæden og fjerner den a-amino-beskyttende gruppe fra sarcosyl-delen og om nødvendigt gentager denne fremgangsmåde for hver sarcosylrest, der skal kobles, og 35 v) fjerner harpiksen og alle beskyttende grupper fra det resulterende peptid med passende reagenser, og om øn- 149091 3 sket fremstiller farmaceutisk acceptable syreadditionssalte af produktet.The process of the invention is characterized in that i) binds a sarcosine or N-protected lysine protected on the α-amino group to a polymeric resin by covalent bonding, which polymer is one containing a functional group, to which the first protected amino acid can be attached by the covalent bond; ii) removing the α-amino protecting group from the amino acid resin and, if necessary, lysine which is N- and N-protected to SAR- the resin and removes the α-amino protecting group from the LYS-SAR resin; iii) couples glutamine or desaminoglutamine protected in its optional α-amino group and in its ε-amino group to the B-amino acid resin; and iv ) when the A group contains one or more sarcosyl moieties, removes the α-amino protecting group from the GLN-B resin and binds sarcosine protected on its α-amino group to the chain and removes the α-amino protecting group from sarcosyl part and if necessary repeats this f and (v) remove the resin and all protective groups from the resulting peptide with appropriate reagents and, if desired, produce pharmaceutically acceptable acid addition salts of the product.

Peptiderne, hvori A betegner en gruppe udvalgt blandt desamino-GLN og GLN, har væsentligt simplere struktur og er væsentligt lettere 05 at fremstille end de før nævnte kendte pentapeptider, eftersom disse materialer blot er dipeptider eller tripeptider. Peptiderne, hvori A er en gruppe udvalgt blandt desamino-GLN og (SAR)n-GLN, hvori n er 2, 3 eller 4 og B er udvalgt fra gruppen bestående af LYS-OH og LYS-NHg, er væsentligt mere aktive end de før nævnte pentapep-10 tider, idet de udviser aktivitet ved den nedenfor beskrevne kyllinge--induktionsprøve ved koncentrationer fra ca. 10 femptogram (fg)/ml til ca. 100 ng/ml. Disse peptider er derfor indtil 10 millioner gange mere kraftige end de før nævnte pentapeptider. Da visse af disse mere kraftige peptider også har meget simpel struktur, forener de i 15 væsentlig grad forøget styrke med forholdsmæssig lettere og mere økonomisk fremstilling.The peptides, wherein A represents a group selected from desamino-GLN and GLN, have substantially simpler structure and are significantly easier to prepare than the previously known pentapeptides, since these materials are merely dipeptides or tripeptides. The peptides wherein A is a group selected from desamino-GLN and (SAR) n-GLN, wherein n is 2, 3 or 4 and B is selected from the group consisting of LYS-OH and LYS-NHg, are significantly more active than the prior to said pentapept times, exhibiting activity in the chicken described below - induction test at concentrations of about 10 femptogram (fg) / ml to approx. 100 ng / ml. Therefore, these peptides are up to 10 million times more potent than the aforementioned pentapeptides. Since some of these more powerful peptides also have very simple structure, they combine substantially increased strength with relatively lighter and more economical production.

Inden for opfindelsens rammer falder endvidere fremstillingen af de farmaceutisk acceptable syreadditionssalte af peptiderne. Som syrer, der er i stand til at danne salte med peptiderne, kan nævnes 20 uorganiske syrer såsom saltsyre, hydrogenbromidsyre, perchlorsyre, salpetersyre, thiocyansyre, svovlsyre, fosforsyre, etc. og organiske syrer såsom myresyre, eddikesyre, propionsyre, glycolsyre, mælkesyre, pyrodruesyre, oxalsyre, malonsyre, ravsyre, maleinsyre, fumar-syre, anthranilsyre, kanelsyre, naphthalensulfonsyre eller sulfanilsyre, 25 fx.Furthermore, within the scope of the invention, the preparation of the pharmaceutically acceptable acid addition salts of the peptides falls. As acids capable of forming salts with the peptides may be mentioned 20 inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, phosphoric, etc. and organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid or sulphanilic acid, e.g.

I de ovennævnte strukturer er peptidernes aminosyrekomponen-ter af nemhedsgrunde identificeret ved forkortelser. Disse forkortelser er følgende: 30 Aminosyre ForkortelseIn the above structures, the amino acid components of the peptides are identified by abbreviations for convenience. These abbreviations are as follows: 30 Amino Acid Abbreviation

L-glutamin GLUL-glutamine GLU

L-alanin ALAL-alanine ALA

L-lysin LYSL-lysine LIGHT

L-tyrosin TYRL-tyrosine TYR

35 L-asparagin ASN35 L-asparagine ASN

L-isoleucin ILEL-isoleucine ILE

Sarcosin SARSarcosin SAR

149091 4149091 4

Udtrykket "desamino-GLN" refererer til en L-glutamin-rest, hvori «-aminogruppen er erstattet af hydrogen, d.v.s. med formlen h2noc-(ch2)3-co-.The term "desamino-GLN" refers to an L-glutamine residue in which the "amino group is replaced by hydrogen, i.e. of the formula h2noc- (ch2) 3 -co-.

De omhandlede peptider indeholder dipeptiddelen -GLN-LYS-05 enten i sig selv eller i kombination med fra én til fem sarcosin-radi-kaler, idet nogle forbindelser mangler α-aminogruppen i en N-terminal glutamylrest. Disse peptider har vist sig at udvise lignende egenskaber som det i USA-patentskrift nr. 4.002.602 omhandlede 74 aminosyrer lange polypeptid, ubiquitin. Peptiderne fremstillet ifølge 10 den foreliggende opfindelse udmærker sig især ved deres evne til at inducere den selektive differentiering af T-prækursor-celler og B-prækursor-celler i så små som femptogram/ml koncentrationer. Udover den overraskende kraftige ubiquitin-lignende aktivitet er det overraskende, at dette dipeptid-fragment, enten alene eller i kombi-15 nation med fra én til fem rester af den ikke naturlige aminosyre sar-cosin besidder nogen aktivitet overhovedet. Betydningen af den foreliggende opfindelse ligger i den enestående kombination af peptidets ekstreme simpelhed og overvældende styrke. Således har den mindst kraftige af de omhandlede peptider (H-GLN-LYS-NHg) den samme 20 styrke som thymopoietin-pentapeptidet, men er blot et dipeptid. Det mest kraftige af de omhandlede peptider (H-SAR-SAR-SAR-GLN-LYS-NHg) et omkring 10 millioner gange mere kraftigt end thymopoietin-pentapeptidet. De her omhandlede peptider har således i koncentrationer så lave som 10 femptogram/ml vist sig at inducere differentie-25 ringen af både T-prækursor-celler målt ved forekomsten af de thy-miske differentierings-antigener TL og THY-I (Θ), og B-prækursor-cefier som målt ved forekomsten af receptorer for komplement, et distinktivt tegn pi B-celler.The present peptides contain the dipeptide moiety -GLN-LYS-05 either on its own or in combination with from one to five sarcosine radicals, some compounds lacking the α-amino group in an N-terminal glutamyl residue. These peptides have been found to exhibit similar properties to the 74 amino acid long polypeptide disclosed in U.S. Patent No. 4,002,602. The peptides prepared according to the present invention are particularly distinguished by their ability to induce the selective differentiation of T precursor cells and B precursor cells at concentrations as low as femptogram / ml. In addition to the surprisingly potent ubiquitin-like activity, it is surprising that this dipeptide fragment, either alone or in combination with from one to five residues of the unnatural amino acid sarcosine, has no activity at all. The significance of the present invention lies in the unique combination of the extreme simplicity and overwhelming potency of the peptide. Thus, the least potent of the subject peptides (H-GLN-LYS-NHg) has the same potency as the thymopoietin pentapeptide, but is merely a dipeptide. The most potent of the subject peptides (H-SAR-SAR-SAR-GLN-LYS-NHg) is about 10 million times more potent than the thymopoietin pentapeptide. Thus, the present peptides have been shown to induce the differentiation of both T precursor cells as measured by the presence of the thymic differentiation antigens TL and THY-I (Θ) at concentrations as low as 10 femptogram / ml. and B precursor cephies as measured by the presence of complement receptors, a distinctive sign of B cells.

For at lette forståelsen af den overraskende styrke af de om-30 handlede peptider gives følgende tabel for måleenheder.To facilitate understanding of the surprising strength of the peptides in question, the following table of units of measurement is given.

Måleenhed (forkortelse) Vægt i gram -3 milligram (mg) 10 *6 mikrogram (pg) 10 -9 35 nanogram (ng) 10 -12 picogram (pg) 10 femptogram (fg) 10 -15 149091 5Unit of measurement (abbreviation) Weight in grams -3 milligrams (mg) 10 * 6 micrograms (pg) 10 -9 35 nanograms (ng) 10 -12 picograms (pg) 10 femptograms (fg) 10 -15 149091 5

Blandt de omhandlede peptider foretrækkes sådanne, hvori A er udvalgt fra gruppen bestående af desamino-GLN og H-(SAR)n~GLN, hvor n er 2, 3 eller 4 og B er udvalgt fra gruppen bestående af LYS-OH og LYS-NHg- Disse foretrukne peptider udviser aktivitet 05 ved koncentrationer i intervallet fra 10 fg/ml til ca. 100 ng/ml.Among the present peptides, those in which A is selected from the group consisting of desamino-GLN and H- (SAR) n ~ GLN are preferred, where n is 2, 3 or 4 and B is selected from the group consisting of LYS-OH and LYS- NHg- These preferred peptides exhibit activity 05 at concentrations ranging from 10 µg / ml to ca. 100 ng / ml.

Peptidsekvensen GLN-LYS-SAR-NHg kan kemisk vises som: H2N-<j:H-CONH-CjH-CON-CH2-CONH2 ΐΗ2 (CH2)4CH3 10 CH0 NH, I 2 2 CONHg H - GLN - LYS - SAR - NHg 15 Blandt de omhandlede peptider foretrækkes endvidere sådanne, som har formlen (desamino-GLN)-LYS-C, hvori C er udvalgt fra gruppen bestående af OH og NHg. Disse foretrukne desaminodipeptider forener overvældende styrke med ekstrem simpelhed i struktur og fremstillingslethed. Disse foretrukne desaminodipeptider er af en så 20 simpel struktur, at de kan krystalliseres, i modsætning til større peptider, der i almindelighed ikke kan. Disse desaminodipeptiders evne til at krystallisere er en overordentlig væsentlig faktor ved simplificeringen af rensningen deraf.The peptide sequence GLN-LYS-SAR-NHg can be chemically shown as: H2N- <j: H-CONH-CjH-CON-CH2-CONH2 ΐΗ2 (CH2) 4CH3 10 CH0 NH, I 2 2 CONHg H - GLN - LYS - SAR - NHg 15 Also preferred are those which have the formula (desamino-GLN) -LYS-C, wherein C is selected from the group consisting of OH and NHg. These preferred desaminodipeptides combine overwhelming strength with extreme simplicity in structure and ease of manufacture. These preferred desaminodipeptides are of such a simple structure that they can be crystallized, unlike larger peptides which generally cannot. The ability of these desaminodipeptides to crystallize is an extremely important factor in the simplification of its purification.

For bedre at forstå vigtigheden af de omhandlede peptiders dif-25 ferentierende biologiske egenskaber, skal det omtales, at thymus' -funktion i relation til immunitet generelt kan angives som produktionen af thymus-afledte celler (lymfocyter), der kaldes T-celler. T-cel-ler udgør en stor del af mængden af recirkulerende små lymfocyter.To better understand the importance of the differentiating biological properties of the peptides of the present invention, it should be mentioned that thymus' function in relation to immunity can generally be referred to as the production of thymus-derived cells (lymphocytes) called T cells. T cells form a large part of the amount of recirculating small lymphocytes.

T-celler har immunologisk specificitet og er direkte involveret i celle-30 medierede immun-reaktioner (såsom homotransplantations-reaktioner), som effektor-celler. T-celler sekreterer imidlertid ikke humorale antistoffer. Disse antistoffer sekreteres af celler (kaldet B-celler), som afledes direkte fra benmarven uafhængigt af den thymiske indflydelse.T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homotransplantation reactions), such as effector cells. However, T cells do not secrete humoral antibodies. These antibodies are secreted by cells (called B cells) that are derived directly from the bone marrow independently of the thymic influence.

For mange antigeners vedkommende kræver B-celler imidlertid tilstede-35 værelsen af passende reaktive T-celler før de kan producere antistoffer. T-celler er også involveret i regulering af immunsystemet som hjælpe-celler, suppressor-celler etc. Mekanismen af denne proces ved celle-samvirke forstis endnu ikke fuldstændigt.However, for many antigens, B cells require the presence of appropriate reactive T cells before they can produce antibodies. T cells are also involved in regulation of the immune system such as auxiliary cells, suppressor cells, etc. The mechanism of this process of cell co-operation is not yet fully understood.

149091 6 På baggrund af denne forklaring kan det operationelt siges, at thymus er nødvendig for udviklingen af cellulær immunitet og mange humorale antistof-reaktioner samt for regulering af immunsystemet.Based on this explanation, it can be operationally said that thymus is necessary for the development of cellular immunity and many humoral antibody responses as well as for regulation of the immune system.

Disse resultater opnås i det mindste delvis ved induktionen, i thy-05 mus, af differentieringen af haemopoietiske stam-celler til T-celler.These results are obtained, at least in part, by the induction, in thy-05 mice, of the differentiation of haemopoietic stem cells into T cells.

Denne induktive påvirkning medieres af sekretioner af thymus' epi-thel-celler, dvs. af thymus-hormonerne.This inductive effect is mediated by secretions of thymus epithelial cells, ie. of the thymus hormones.

Endvidere skal det til forståelse af virkningen af thymus og lymfocyternes cellesystem samt lymfocyternes cirkulation i legemet, 10 anføres at stamceller opstår i benmarven og når thymus via blodbanerne. Inde i thymus bliver stamceller differentieret til immunologisk kompetente T-celler, der migrerer til blodbanerne og sammen med B-celler cirkulerer mellem væv, lymfekar og blodbaner.Furthermore, in order to understand the effect of the thymus and the lymphocyte cell system as well as the circulation of the lymphocytes in the body, it should be stated that stem cells arise in the bone marrow and reach the thymus via the bloodstream. Inside the thymus, stem cells are differentiated into immunologically competent T cells that migrate to the bloodstream and, together with B cells, circulate between tissues, lymph vessels, and bloodstreams.

Legemets celler, som udskiller antistof (B-cellerne) udvikles 15 også fra haemopoietiske stamceller, men deres differentiering bestemmes ikke af thymus. I fugle differentieres de i et med thymus analogt organ, der kaldes Bursa Fabricii. I pattedyr har man ikke fundet et ækvivalent organ og det antages, at B-celler differentierer i benmarven. De kaldes derfor benmarvs-afledte celler eller B-celler.The body's cells that secrete antibody (B cells) are also developed from haemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they are differentiated into a thymus analogous organ called Bursa Fabricii. In mammals, no equivalent organ has been found and B cells are believed to differentiate into the bone marrow. They are therefore called bone marrow-derived cells or B cells.

20 De fysiologiske stoffer, som dikterer denne differentiering, er totalt ukendte.20 The physiological substances that dictate this differentiation are totally unknown.

Som anført ovenfor, er de her omhandlede peptider terapeutisk værdifulde ved behandlingen af mennesker og dyr. Da de hidtil ukendte peptider er i stand til at inducere differentieringen af lymfopoieti-25 ske stamceller, som opstår i det haemopoietiske væv, til både thymus-afledte lymfocyter (T-celler) og immunokompetente B-celler, som er i stand til at indgå i legemets immunreaktion, antages de ifølge opfindelsen tilvejebragte produkter at have multiple terapeutiske anvendelser. Da forbindelserne er i stand til at udføre visse af de angiv-30 ne funktioner af thymus, har de primært anvendelse inden for forskellige thymus-funktions- og immunitets-områder. Et primært anvendelsesområde er ved behandlingen af DiGeorge syndrom, en tilstand, hvor der er et medfødt fravær af thymus. Injektion af poly-peptiderne vil afhjælpe denne mangel. En anden anvendelse er ved 35 agammaglobulinæmia, der skyldes en defekt hos legemets formodede B-celle differentierende hormon. Injektion af polypeptiderne vil afhjælpe denne defekt. Da peptiderne er aktive ved meget lave kon- 149091 7 centrationer, er de værdifulde til at assistere ved legemets kollektive immunitet ved at de forøger eller assisterer med terapeutisk stimulering af cellulær immunitet og humoral immunitet, hvorved de bliver værdifulde til behandling af sygdomme, der involverer kronisk 05 infektion in vivo såsom fungale eller mycoplasma infektioner, tuberkulose, spedalskhed, akute og kroniske virale infektioner o.I. Endvidere antages peptiderne at være nyttige inden for ethvert område, hvor cellulær eller humoral immunitet har betydning og især, hvor der er immunitetsmangler såsom ved det ovennævnte DiGeorge syn-10 drom. Endvidere har peptiderne på grund af deres egenskaber in vitro anvendelighed til inducering af udviklingen af overflade-antigener af T-celler, til inducering af udviklingen af den funktionelte evne til at opnå reaktion på mitogener og antigener samt celle-samvirke ved at forbedre B-cellernes evne til at producere antistoffer. De 15 har in vitro anvendelighed ved at inducere udviklingen af B-celler som målt ved udviklingen af overfladereceptorer for komplement. Peptiderne er også nyttige til inhibering af den u kontrol lerede formering af lymfocyter, som reagerer over for ubiquitin (USA-patentskrift nr. 4.002.602). En vigtig egenskab ved peptiderne ligger i deres in 20 vivo evne til at genopbygge celler med T-cellernes egenskaber samt i deres in vivo evne til at genopbygge celler med B-cellernes egenskaber. De er derfor også nyttige til behandling af relative eller absolute B-celle-mangler samt relative eller absolute T-celle-mangler, uanset om disse mangler skyldes mangler i vævet, som differentierer 25 B-celler, hhv. i thymus eller har en helt anden årsag.As stated above, the peptides of this invention are therapeutically valuable in the treatment of humans and animals. Since the novel peptides are capable of inducing the differentiation of lymphopoietic stem cells arising in the haemopoietic tissue to both thymus-derived lymphocytes (T cells) and immunocompetent B cells capable of in the body's immune response, the products provided by the invention are believed to have multiple therapeutic uses. Since the compounds are capable of performing certain of the stated functions of the thymus, they are of primary use in various fields of function and immunity. A primary area of application is in the treatment of DiGeorge syndrome, a condition in which there is an innate absence of thymus. Injection of the polypeptides will alleviate this deficiency. Another use is in 35 agammaglobulinemia due to a defect in the body's putative B-cell differentiating hormone. Injection of the polypeptides will alleviate this defect. As the peptides are active at very low concentrations, they are valuable in assisting the body's collective immunity by enhancing or assisting with therapeutic stimulation of cellular immunity and humoral immunity, thereby becoming valuable in the treatment of diseases involving chronic 05 infection in vivo such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and Furthermore, the peptides are believed to be useful in any area where cellular or humoral immunity is important and especially where there are immune deficiencies such as in the above DiGeorge syndrome. Furthermore, because of their in vitro properties, the peptides have utility in inducing the development of surface antigens of T cells, in inducing the development of the functional ability to respond to mitogens and antigens, as well as in cell cooperation by enhancing the B cells. ability to produce antibodies. The 15 have in vitro utility in inducing the development of B cells as measured by the development of complement surface receptors. The peptides are also useful for inhibiting the uncontrolled proliferation of lymphocytes responsive to ubiquitin (U.S. Patent No. 4,002,602). An important property of the peptides lies in their in vivo ability to rebuild cells with the characteristics of T cells as well as in their in vivo ability to rebuild cells with the characteristics of B cells. Therefore, they are also useful for treating relative or absolute B-cell deficiencies as well as relative or absolute T-cell deficiencies, whether these deficiencies are due to deficiencies in the tissue that differentiate 25 B cells, respectively. in the thymus or has a completely different cause.

En yderligere vigtig egenskab ved de ifølge opfindelsen tilvejebragte peptider er at de er meget aktive i meget lave koncentrationer.A further important feature of the peptides provided by the invention is that they are very active at very low concentrations.

Det har således vist sig, at peptiderne i almindelighed er aktive i koncentrationer fra ca. 10 pg/ml til 10 ng/ml og de foretrukne pep-30 tider er aktive ved koncentrationer varierende fra ca. 10 fg/ml.Thus, it has been found that the peptides are generally active at concentrations ranging from ca. 10 µg / ml to 10 ng / ml, and the preferred peptides are active at concentrations ranging from ca. 10 µg / ml.

Bæreren kan være enhver af de velkendte bærere til dette formål, herunder normale saltopløsninger, fortrinsvis med en protein-fortynder såsom okseserumalbumin for at hindre adsorptionstab til glasapparatur ved disse lave koncentrationer. De omhandlede peptider 35 er i almindelighed aktive i et område fra over ca. 1 ng/kg legemsvægt og de foretrukne peptider er aktive fra ca. 10 pg/kg. Til behandling af DiGeorge syndrom kan peptiderne administreres i en mængde på ca. 1 til ca. 100 ng/kg legemsvægt. I almindelighed kan 149091 8 det samme område af dosismængder anvendes til behandling af de øvrige omtalte tilstande eller sygdomme. Uanset at den foregående omtale har vedrørt parenteral administrering, skal det forstås, at oral administrering også er mulig i dosismængder, der i almindelighed 05 er ca. 100 til 1000 gange større end ved injektion. Andre velkendte administrationsveje, fx. sublinguale, rektale, nasale, etc. kan også anvendes.The carrier may be any of the well known carriers for this purpose, including normal saline solutions, preferably with a protein diluent such as bovine serum albumin to prevent loss of adsorption to glass apparatus at these low concentrations. The present peptides 35 are generally active in a range of more than ca. 1 ng / kg body weight and the preferred peptides are active from ca. 10 pg / kg. For the treatment of DiGeorge syndrome, the peptides can be administered in an amount of approx. 1 to approx. 100 ng / kg body weight. In general, the same range of dosage amounts can be used to treat the other conditions or diseases mentioned. Although the foregoing mention has related to parenteral administration, it should be understood that oral administration is also possible in dosage doses which in general 05 are approx. 100 to 1000 times greater than injection. Other well-known routes of administration, e.g. sublingual, rectal, nasal, etc. may also be used.

Til fremstilling af farmaceutiske præparater kombineres et peptid med formel I eller et syreadditionssalt deraf som aktiv bestanddel i 10 intim blanding med en farmaceutisk bærer iht. konventionel farmaceutisk præparationsteknik, hvilken bærer kan antage mange forskellige former i afhængighed af den til administrering ønskede præparatform, fx. sublingual, rektal, nasal, oral eller parenteral. Til fremstilling af præparaterne på oral dosisform kan ethvert af de sædvanlige far-15 maceutiske medier anvendes, som fx. vand, glykoler, olier, alkoholer, smagsstoffer, præserveringsmidler, farvestoffer o.I. i tilfælde af orale flydende præparater som fx. suspensioner, eleksirer og opløsninger, eller bærere såsom stivelser, sukkerarter, fortyndingsmidler, granuleringsmidler, smøremidler, bindemidler, disintegreringsmidler 20 o.I. i tilfælde af orale faste præparater som fx. pulvere, kapsler og tabletter. P.g.a. deres administreringslethed repræsenterer tabletter og kapsler den mest fordelagtige orale enhedsdosisform, i hvilket tilfælde der naturligvis anvendes faste farmaceutiske bærere. Om ønsket kan tabletter overtrækkes med sukker eller enterisk ved hjælp 25 af standardmetoder. Til parenterale præparater vil bæreren sædvanligvis omfatte sterilt vand, selvom andre bestanddele, fx. for at lette opiøseligheden eller til præserveringsformål, kan tilsættes. Injicerba-re suspensioner kan også fremstilles, i hvilket tilfælde der anvendes passende flydende bærere, suspensionsmidler o.I. De parenterale 30 farmaceutiske præparater bør udformes til administrering af de omhandlede peptider i en mængde på ca. 10 pg/kg til ca. 100 ng/kg legemsvægt. De orale præparater bør kunne administreres ca. 100 til 1000 gange dosen for parenteral administrering, dvs. fra ca. 1 ng/kg til ca. 100 pg/kg legemsvægt. Følgelig bør de parenterale præ-35 parater per dosisenhed indeholde fra ca. 500 pg til ca. 5 pg, mens de orale præparater per enhedsdosis bør indeholde fra ca. 50 ng til ca. 5 mg af det omhandlede peptid.For the preparation of pharmaceutical compositions, a peptide of formula I or an acid addition salt thereof is combined as an active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical preparation technique, which carrier can take many different forms depending on the formulation desired for administration, e.g. sublingual, rectal, nasal, oral or parenteral. For the preparation of the compositions in oral dosage form, any of the usual pharmaceutical media may be used, e.g. water, glycols, oils, alcohols, flavors, preservatives, dyes and the like. in the case of oral liquid preparations such as e.g. suspensions, elixirs and solutions, or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents 20 o.I. in the case of oral solid preparations such as e.g. powders, capsules and tablets. P.g.a. their ease of administration, tablets and capsules represent the most advantageous oral unit dosage form, in which case solid pharmaceutical carriers are naturally used. If desired, tablets may be coated with sugar or enteric by standard methods. For parenteral preparations, the carrier will usually comprise sterile water, although other ingredients, e.g. for ease of dissolvability or for preservation purposes may be added. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like are used. The parenteral 30 pharmaceutical compositions should be designed to administer the subject peptides in an amount of about 10 pg / kg to approx. 100 ng / kg body weight. The oral preparations should be capable of being administered approx. 100 to 1000 times the dose for parenteral administration, i.e. from approx. 1 ng / kg to approx. 100 pg / kg body weight. Accordingly, the parenteral preparations per unit dose should comprise from ca. 500 pg to approx. 5 µg, while the oral preparations per unit dose should contain from approx. 50 ng to approx. 5 mg of the subject peptide.

149091 9149091 9

De omhandlede peptider blev fremstillet under anvendelse af lignende principper som beskrevet af Merrifield i Journal of American Chemical Society, 85, s. 2149-2154, 1963. Syntesen involverede den trinvise tilsætning af beskyttede aminosyrer til en voksende peptid-05 kæde, der var bundet med covalente bindinger til en fast harpikspartikel. Ved denne fremgangsmåde blev reagenser og biprodukter fjernet ved filtrering og rensning af mellemprodukter blev elimineret.The present peptides were prepared using similar principles as described by Merrifield in the Journal of the American Chemical Society, 85, pp. 2149-2154, 1963. The synthesis involved the stepwise addition of protected amino acids to a growing peptide-05 chain bound with covalent bonds to a solid resin particle. In this process, reagents and by-products were removed by filtration and purification of intermediates was eliminated.

Det almene princip ved denne fremgangsmåde beror på fastgørelse af kædens C-terminale aminosyre til en fast polymer ved hjælp af en 10 covalent binding og tilsætning af de efterfølgende aminosyrer én ad gangen på trinvis måde indtil den ønskede sekvens er samlet. Endelig fjernes peptidet fra den faste bærer og beskyttende grupper fjernes. Denne fremgangsmåde giver en voksende peptid kæde, som er fastgjort til en fuldstændig uopløselig fast partikel, således at 15 den er på en bekvem form til filtrering og udvaskning for reagenser og biprodukter.The general principle of this method is the attachment of the chain's C-terminal amino acid to a solid polymer by a covalent bond and addition of the subsequent amino acids one step at a time until the desired sequence is assembled. Finally, the peptide is removed from the solid support and protective groups are removed. This process provides a growing peptide chain attached to a completely insoluble solid particle such that it is in a convenient form for filtration and leaching of reagents and by-products.

Aminosyrerne kan fastgøres til enhver egnet polymer, der blot skal være let adskillelig fra de uomsatte reagenser. Polymeren kan være uopløselig i de anvendte opløsningsmidler eller kan være opløse-20 lig i visse opløsningsmidler og uopløselig i andre. Polymeren bør have en stabil fysisk form, som tillader let filtrering. Den skal indeholde en funktionel gruppe, hvortil den første beskyttede aminosyre kan bindes fast ved hjælp af den covalente binding. Forskellige uopløselige polymere, som er egnede til dette formål, er fx. cellulose, 25 polyvinylalkohol, polymethacrylat og sulfoneret polystyren, men ved syntesen ifølge den foreliggende opfindelse anvendtes en chlormethy-leret copolymer af styren og divinylbenzen. Polymere, som er opløselige i organiske opløsningsmidler, men uopløselige i vandige opløsningsmidler, kan også anvendes. En sådan polymer er en polyethy-30 len/ glycol med en molekylvægt på ca. 20.000, der er opløselig i me-thylenchlorid, men uopløselig i vand. Anvendelsen af denne polymer ved peptidsyntese er beskrevet i F. Bayer og M. Mutter, Nature, 237, 512 (1972) samt de deri angivne referencer.The amino acids can be attached to any suitable polymer which should be readily separable from the unreacted reagents. The polymer may be insoluble in the solvents used or may be soluble in some solvents and insoluble in others. The polymer should have a stable physical form which allows easy filtration. It must contain a functional group to which the first protected amino acid can be attached by the covalent bond. Various insoluble polymers suitable for this purpose are e.g. cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene, but in the synthesis of the present invention a chloromethylated copolymer of styrene and divinylbenzene was used. Polymers which are soluble in organic solvents but insoluble in aqueous solvents may also be used. One such polymer is a polyethylene / glycol having a molecular weight of approx. 20,000, which is soluble in methylene chloride but insoluble in water. The use of this polymer in peptide synthesis is described in F. Bayer and M. Mutter, Nature, 237, 512 (1972) as well as the references cited therein.

De forskellige funktionelle grupper på aminosyren, som var ak-35 tive, men som ikke skulle indgå i reaktionerne, beskyttedes under hele reaktionen ved hjælp af konventionelle beskyttende grupper, som anvendt inden for polypeptidteknikken. Således beskyttedes den 149091 10 funktionelle gruppe pi lysin med beskyttende grupper, der kunne fjernes ved sekvensens afslutning uden ugunstigt at påvirke det endelige polypeptidprodukt. I. syntesen anvendtes ninhydrin til bestemmelse af, om koblingen var komplet. Såfremt der ikke påvistes 05 komplet kobling, gentoges koblingen med den samme beskyttede aminosyre før afbeskyttelse.The various functional groups on the amino acid which were active but which were not to be included in the reactions were protected throughout the reaction by conventional protecting groups as used in the polypeptide technique. Thus, the functional group pi lysine was protected with protective groups which could be removed at the end of the sequence without adversely affecting the final polypeptide product. In the synthesis, ninhydrin was used to determine whether the coupling was complete. If no complete coupling was detected, the coupling was repeated with the same protected amino acid before deprotection.

Den C-terminale aminosyre kan fastgøres til polymeren på en række velkendte måder. Resumeer af fremgangsmåder til fastgørelse til halomethylharpikser er givet i Horiki, et al., Chem. Letters, s.The C-terminal amino acid can be attached to the polymer in a number of well-known ways. Summaries of methods for attachment to halomethyl resins are given in Horiki, et al., Chem. Letters, p.

10 165-168 (1978) og Gisin, Helv. Chim. Acta, 56, 1476 (1973) samt de deri anførte referencer. Såfremt der skal fremstilles et C-terminalt amid, kan en af to veje anvendes. Enten kan den iht. Merrifield-tek-nikken fremstillede peptid-harpiks spaltes fra harpiksen under anvendelse af vandfri ammoniak, eller en benzhydrylamin-harpiks kan an-15 vendes. Spaltning fra sidstnævnte harpiks med hydrogenfluorid giver det C-terminale amid-peptid. Anvendelse af en benzhydrylamin-harpiks.er fx. vist i J. Rivier, et al., J. Med. Chem., 16, s. 545-549 (1973).10 165-168 (1978) and Gisin, Helv. Chim. Acta, 56, 1476 (1973) and the references cited therein. If a C-terminal amide is to be prepared, one of two pathways can be used. Either the peptide resin prepared according to the Merrifield technique can be cleaved from the resin using anhydrous ammonia or a benzhydrylamine resin can be used. Cleavage from the latter resin with hydrogen fluoride gives the C-terminal amide peptide. Use of a benzhydrylamine resin is e.g. shown in J. Rivier, et al., J. Med. Chem., 16, pp. 545-549 (1973).

Den almene fremgangsmåde til fremstilling af C-terminal carboxyl-20 peptider involverede først esterificering af L-lysin, beskyttet på dens aminogrupper, til chformethylharpiksen. ved hjælp af den i ovennævnte Gisin-artikel omtalte CsHCOg-metode. Den beskyttende gruppe på a-aminogruppen af lysin-aminosyren (f.eks. t-BOC, dvs. t-butyloxycarbonyl) fjernedes dernæst uden påvirkning af andre be-25 skyttende grupper. Den koblede aminosyre^harpiks filtreredes dernæst, vaskedes og neutraliseredes. Den resulterende'koblede amino-syre-harpiks med den frie aminogruppe omsattes dernæst med en beskyttet L-glutamin, fortrinsvis o^t-BOC-L-glutamin til kobling af L-glutaminen. Sekvensen af reaktioner udførtes som følger: 30 35 U9091 11The general process for preparing C-terminal carboxyl peptides first involved esterification of L-lysine, protected on its amino groups, to the chformethyl resin. using the CsHCOg method mentioned in the above Gisin article. The protecting group on the α-amino group of the lysine amino acid (eg t-BOC, i.e. t-butyloxycarbonyl) was then removed without the influence of other protecting groups. The coupled amino acid resin was then filtered, washed and neutralized. The resulting free amino group coupled amino acid resin was then reacted with a protected L-glutamine, preferably o-t-BOC-L-glutamine, to link the L-glutamine. The sequence of reactions was carried out as follows:

Harpiks flResin fl

a-R2-Lys-COOHa-Lys-R2-COOH

fl 05 a-R?-Lys-harpiks \ Fjern a-amino beskyttende gruppe .fl 05 α-R? -Lysin resin \ Remove α-amino protecting group.

H-L^s-harpiks 10 a-Rg-GIn l1 a-R2-Gln-Lys-harpiksH-L ^ s-Resin 10a-Rg-GIn 11a-R2-Gln-Lys Resin

Fjern alle beskyttende grupper og harpiksRemove all protective groups and resins

15 H-Glh-Lys-OHH-Glh-Lys-OH

I ovenstående reaktionssekvens betegner R1 en beskyttende gruppe på den reaktionsdygtige sidekæde pi L-lysin, som ikke påvirkes eller fjernes, når den beskyttende gruppe pi ot-aminogrup-20 pen fjernes for at tillade yderligere omsætning, og a-R2 er en beskyttende gruppe på α-aminogruppen. Fortrinsvis betegner i ovenstående pentapeptid-harpiks mellemprodukt, R^ en beskyttende gruppe, såsom 2-chlorbenzyloxycarbonyl, og R2 betegner t-butyloxycar-bonyl. Harpiksen er en vilkårlig blandt de ovenfor omtalte harpikser, 25 som kan anvendes til fremgangsmåden.In the above reaction sequence, R1 represents a protecting group on the reactive side chain p1 L-lysine which is not affected or removed when the protecting group p1 o-amino group is removed to allow further reaction and α-R2 is a protecting group of α-amino group. Preferably, in the above pentapeptide resin intermediate, R 1 represents a protecting group such as 2-chlorobenzyloxycarbonyl, and R 2 represents t-butyloxycarbonyl. The resin is any of the resins mentioned above which can be used in the process.

Når det sidste mellemprodukt er fremstillet, spaltes peptidharpiksen til fjernelse af de beskyttende grupper R^ og R2 derpå samt harpiksen. De beskyttende grupper fjernes på konventionel måde, f.eks. ved behandling med vandfri hydrogenfluorid, og det resulte-30 rende frie peptid udvindes dernæst.When the last intermediate is prepared, the peptide resin is cleaved to remove the protecting groups R 1 and R 2 thereon as well as the resin. The protecting groups are removed in a conventional manner, e.g. by treatment with anhydrous hydrogen fluoride and the resulting free peptide is then recovered.

Som anført ovenfor er det ved udøvelsen af fremgangsmåden nødvendigt at beskytte eller blokere aminogrupperne for at kontrol- . lere reaktionen og opnå de ønskede produkter. Egnede aminobeskyt-tende grupper, som kan anvendes, omfatter saltdannelse til beskyt-35 telse af stærkt basiske aminogrupper, eller urethan-beskyttende substitutter, såsom benzyloxycarbonyl og t-butyloxycårbonyl·.· Det foretrækkes at anvende t-butyloxycarbonyl (t-BOC) eller t-amyloxycar- 149091 12 bonyl (AOC) til beskyttelse af α-aminogruppen i de aminosyrer, som undergår reaktion ved molekylets carboxylende, eftersom BOC og AOC (t-amyloxycarbonyl) som beskyttende grupper let fjernes efter en sådan reaktion og inden det efterfølgende trin (hvor en sådan 05 α-aminogruppe selv undergår reaktion) ved relativ mild indvirkning af syrer, (f.eks. trifluoreddikesyre), hvilken behandling ikke iøv-rigt påvirker grupper, som anvendes til at beskytte andre reaktive sidekæder. Det vil således kunne forstås, at a-aminogrupperne kan beskyttes ved omsætning med ethvert materiale, der vil beskytte 70 aminogrupperne for den eller de efterfølgende reaktioner, men som senere kan fjernes under betingelser, som ikke iøvrigt vil påvirke molekylet. Eksempler på sådanne materialer er organiske carboxylsyre- eller carbonsyrederivater, der vil acylere aminogruppen.As stated above, in the practice of the process, it is necessary to protect or block the amino groups in order to control. the reaction and obtain the desired products. Suitable amino protecting groups which may be used include salt formation to protect highly basic amino groups, or urethane protecting substitutes such as benzyloxycarbonyl and t-butyloxycarbonyl · It is preferred to use t-butyloxycarbonyl (t-BOC) or t-amyloxycarbonyl (AOC) to protect the α-amino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since BOC and AOC (t-amyloxycarbonyl) as protecting groups are readily removed after such reaction and before the subsequent step (where such 05 α-amino group itself undergoes reaction) by relatively mild action of acids, (e.g., trifluoroacetic acid), which treatment does not otherwise affect groups used to protect other reactive side chains. Thus, it will be appreciated that the α-amino groups can be protected by reaction with any material which will protect the 70 amino groups for the subsequent reaction (s), but which can later be removed under conditions which will not otherwise affect the molecule. Examples of such materials are organic carboxylic acid or carboxylic acid derivatives which will acylate the amino group.

I almindelighed kan enhver af aminogrupperne beskyttes ved 75 omsætning med en forbindelse indeholdende en gruppe med formlenIn general, any of the amino groups can be protected by reaction with a compound containing a group of the formula

OISLAND

R3-°-é- 20 hvori R3 betegner enhver sådan gruppe, som vil hindre aminogruppen i at indgå efterfølgende koblingsreaktioner, og som kan fjernes uden ødelæggelse af molekylet. Således kan være en ligekædet eller forgrenet alkylgruppe, som kan være umættet, fortrinsvis med 1-10 carbonatomer, og fortrinsvis halogen- eller cyano-substitueret, 25 a ry I, fortrinsvis med 6-14 carbonatomer, cycloalkyl, fortrinsvis med 5-8 carbonatomer, aralkyl, fortrinsvis med 7-18 carbonatomer, alka-ryl, fortrinsvis med 7-18 carbonatomer, eller heterocyclyI, f.eks. isonicotinyl. Aryl-, aralkyl- og alkarylgrupperne kan også være yderligere substitueret, såsom med en eller flere alkylgrupper med 1 til 30 ca. 4 carbonatomer. Foretrukne grupper for R3 omfatter t-butyl, t-amyl, phenyl, toly I, xylyl og benzyl. Særligt foretrukne specifikke aminobeskyttende grupper omfatter benzyloxycarbonyl, substitueret benzyloxycarbonyl, hvori phenylringen er substitueret med et eller flere halogener, f.eks. Cl eller Br, nitro, lavere-alkoxy, f.eks., me-35 thoxy eller lavere-alkyl, t-butyloxycarbonyl, t-amyloxycarbonyl, cyc-lohexyloxycarbonyl, vinyloxycarbonyl, adamantyloxycarbonyI, biphenyl isopropoxycarbonyl, og lignende. Andre beskyttende grupper, 13 1A9091 som kan anvendes, omfatter isonicotinyloxycarbonyl, phthaloyl, p-to-lylsulfonyl, formyl, og lignende.Wherein R3 represents any such group which will prevent the amino group from engaging in subsequent coupling reactions and which can be removed without destroying the molecule. Thus, a straight or branched alkyl group which may be unsaturated may be preferably with 1-10 carbon atoms and preferably halogen or cyano-substituted 25 a ry I, preferably with 6-14 carbon atoms, cycloalkyl, preferably with 5-8 carbon atoms. , aralkyl, preferably with 7-18 carbon atoms, alkaryl, preferably with 7-18 carbon atoms, or heterocyclyl, e.g. isonicotinyl. The aryl, aralkyl and alkaryl groups may also be further substituted, such as with one or more alkyl groups having from 1 to 30 approx. 4 carbon atoms. Preferred groups for R 3 include t-butyl, t-amyl, phenyl, toly I, xylyl and benzyl. Particularly preferred specific amino protecting groups include benzyloxycarbonyl, substituted benzyloxycarbonyl, wherein the phenyl ring is substituted by one or more halogens, e.g. C1 or Br, nitro, lower alkoxy, e.g., methoxy or lower alkyl, t-butyloxycarbonyl, t-amyloxycarbonyl, cyclohexyloxycarbonyl, vinyloxycarbonyl, adamantyloxycarbonyl, biphenyl isopropoxycarbonyl, and the like. Other protecting groups which may be used include isonicotinyloxycarbonyl, phthaloyl, p-tolylsulfonyl, formyl, and the like.

Ved udførelse af den almene fremgangsmåde ifølge opfindelsen opbygges peptidet ved omsætning af den frie α-aminogruppe med en 05 forbindelse, som besidder beskyttede aminogrupper. Til omsætning eller kobling bliver forbindelsen, som skal fastgøres, aktiveret ved sin carboxylgruppe, således at carboxylgruppen dernæst kan reagere med den frie ot-aminogruppe på den fastgjorte peptidkæde. For at opnå aktivering kan carboxylgruppen omdannes til enhver reaktiv ΊΟ gruppe, såsom en ester, et anhydrid, et azid, et syrechlorid, eller lignende. Alternativt kan et passende koblingsreagens tilsættes under reaktionen. Egnede koblingsreagenser er omhandlet, f.eks. i Bodanszky et al. Peptide Synthesis, Interscience, 2. udgave, 1976, kapitel 5, herunder carbodiimider (f.eks. dicyclohexylcarbodiimid), 15 carbonyldiimidizol, og lignende.In carrying out the general process of the invention, the peptide is built up by reacting the free α-amino group with a compound having protected amino groups. For reaction or coupling, the compound to be attached is activated by its carboxyl group, so that the carboxyl group can then react with the free o-amino group on the attached peptide chain. To achieve activation, the carboxyl group can be converted to any reactive ΊΟ group such as an ester, anhydride, azide, acid chloride, or the like. Alternatively, a suitable coupling reagent may be added during the reaction. Suitable coupling reagents are provided, e.g. in Bodanszky et al. Peptide Synthesis, Interscience, 2nd Edition, 1976, Chapter 5, including carbodiimides (e.g., dicyclohexylcarbodiimide), carbonyldiimidizole, and the like.

Det skal endvidere forstis, at aminosyredelene under disse re- -aktioner indeholder både aminogrupper og carboxylgrupper, og sædvanligvis indgår den ene gruppe i reaktionen, medens den anden er beskyttet. Før koblingstrinet fjernes den beskyttende gruppe på 20 det fastgjorte peptids a- eller terminale aminogruppe under betingelser, som ikke i væsentlig grad vil påvirke andre beskyttende grupper, f.eks. på lysin-molekylets ε-amino. Den foretrukne fremgangsmåde til udførelse af dette trin er mild acidolyse, såsom ved omsætning ved stuetemperatur med trifluoreddikesyre.Furthermore, it should be understood that during these reactions the amino acid moieties contain both amino groups and carboxyl groups, and usually one group is included in the reaction while the other is protected. Prior to the coupling step, the α-terminal or terminal amino group of the attached peptide is removed under conditions that will not significantly affect other protective groups, e.g. on the ε-amino of the lysine molecule. The preferred method for performing this step is mild acidolysis, such as by reacting at room temperature with trifluoroacetic acid.

25 Som det vil kunne indses, resulterer den ovenfor beskrevne række procestrin i fremstillingen af det specifikke peptid med formlenAs will be appreciated, the above-described process step results in the preparation of the specific peptide of the formula

II H-GLN-LYS-OHII H-GLN-LIGHT-OH

30 Fremstilling af de tilsvarende sarcosin-holdige peptider kan ske ved hjælp af samme reaktionssekvens som ovenfor, hvorved beskyttet sarcosin tilsættes på det eller de passende steder af proceduren.Preparation of the corresponding sarcosine-containing peptides may be carried out by the same reaction sequence as above, whereby protected sarcosine is added at the appropriate site (s) of the procedure.

På denne måde kan f. eks. fremstilles peptider med følgende formler: H-SAR-GLN-LYS-OH, H-SAR-SAR-GLN-LYS-OH, H-SAR-SAR-SAR-35 GLN-LYS-OH, H-SAR-SAR-SAR-SAR-GLN-LYS-OH og H-GLN-LYS- SAR-OH. Peptider med formel I, hvori A betegner desamino-GLN kan fremstilles ved i de ovenfor beskrevne fremgangsmåder at erstatte 149091 14 GLN med beskyttet desamino-GLN. Herved kan fremstilles desamino-GLN-LYS-OH og desamino-GLN-LYS-SAR-OH.In this way, for example, peptides of the following formulas can be prepared: H-SAR-GLN-LYS-OH, H-SAR-SAR-GLN-LYS-OH, H-SAR-SAR-SAR-35 GLN-LYS-OH , H-SAR-SAR-SAR-SAR-GLN-LYS-OH and H-GLN-LYS-SAR-OH. Peptides of formula I wherein A represents desamino-GLN can be prepared by replacing GLN with protected desamino-GLN in the methods described above. Thereby, desamino-GLN-LYS-OH and desamino-GLN-LYS-SAR-OH can be prepared.

Peptider med formel I, hvori B betegner LYS-NHg eller LYS-SAR-NH2 kan fremstilles ved hjælp af den ovenfor beskrevne frem-05 gangsmåde, men med en benzhydrylamin-harpiks i stedet for den der anvendte chlormethylharpiks. På denne måde kan fremstilles amider til de ovenfor beskrevne peptider. En alternativ fremgangsmåde til fremstilling af disse amider består i at spalte fra chlor-methylharpiksen under anvendelse af vandfri ammoniak.Peptides of formula I wherein B represents LYS-NHg or LYS-SAR-NH2 can be prepared by the procedure described above, but with a benzhydrylamine resin instead of the chloromethyl resin used. In this way, amides can be prepared for the peptides described above. An alternative method for preparing these amides consists of cleaving from the chloromethyl resin using anhydrous ammonia.

TO Identiteten, renheden og sekvensen af de omhandlede peptider bestemtes ved hjælp af velkendt teknik i form af tyndtlagskromatogra-fi, elektroforese, aminosyre-analyse, kernemagnetisk resonansspektro-skopi, elementæranalyse, infrarødspektroskopi o.I.TO The identity, purity and sequence of the peptides in question were determined by well-known techniques in the form of thin layer chromatography, electrophoresis, amino acid analysis, nuclear magnetic resonance spectroscopy, elemental analysis, infrared spectroscopy and the like.

Opfindelsen belyses nærmere i de følgende eksempler. Her og i 15 det foregående er alle dele vægtdele med mindre andet er anført.The invention is further illustrated in the following examples. Here and in the foregoing, all parts are by weight unless otherwise stated.

Eksempel 1Example 1

Til fremstilling af ét af de omhandlede peptider indkøbtes følgende materialer kommercielt.For the preparation of one of the present peptides, the following materials were purchased commercially.

2020

Alpha-BOC-L-GlutaminAlpha-BOC-L-Glutamine

Alpha-BOC-s-benzyloxycarbonyl-L-lysin.Alpha-BOC-S-benzyloxycarbonyl-L-lysine.

I disse reagenser betyder BOC t-butyloxycarbonyl. Reagenser 25 af "sequenal"-kvalitet til aminosyresekvensbestemmelse, dicyclohexyl-carbodiimid (DCC), ninhydrin og harpiksen indkøbtes kommercielt.In these reagents, BOC means t-butyloxycarbonyl. "Sequenal" grade reagents for amino acid sequencing, dicyclohexylcarbodiimide (DCC), ninhydrin and the resin were commercially purchased.

Den anvendte harpiks var en benzhydrylamin-harpiks med en kapacitet på 0,5 mækv/g harpiks.The resin used was a benzhydrylamine resin having a capacity of 0.5 meq / g resin.

Til fremstilling af peptidet kobledes a-BOC-e-benzyloxycarbonyl-30 L-lysin til benzhydrylamin-harpiksen under anvendelse af DCC som koblingsreagenset. Den resulterende beskyttede aminosyreharpiks indeholdt 0,4-0,5 mmol aminosyre per gram harpiks. Under anvendelse af en "Schwarz/Mann Automatic Peptide Synthesizer" anvendtes følgende program til kobling af a-BOC-L-glutaminaminosyre til a-BOC-35 ε-benzyloxycarbonyl-L-lysinharpiksen: 14909 1 15 1. Forvaskning med 40% trifluoreddikesyre (TFA) i CH2CI2, én gang, 1,5 minutter.To prepare the peptide, α-BOC-e-benzyloxycarbonyl-30 L-lysine was coupled to the benzhydrylamine resin using DCC as the coupling reagent. The resulting protected amino acid resin contained 0.4-0.5 mmol amino acid per gram of resin. Using a "Schwarz / Mann Automatic Peptide Synthesizer", the following program was used to link α-BOC-L-glutamic amino acid to α-BOC-35 ε-benzyloxycarbonyl-L-lysine resin: 14909 1 15 1. Pre-washing with 40% trifluoroacetic acid ( TFA) in CH 2 Cl 2, once, 1.5 minutes.

2. Afbeskyttelse med 40% TFA i CHgClg, én gang, 20 minut ter.2. Deprotection with 40% TFA in CHgClg, once, 20 minutes ter.

05 3. Vaskning med CHCIg, én gang, 1,5 minutter.05 3. Washing with CHCl 3, once, 1.5 minutes.

4. Vaskning med EtOH, én gang, 1,5 minutter.4. Wash with EtOH, once, 1.5 minutes.

5. Vaskning med CH2CI2, to gange, 1,5 minutter.5. Wash with CH 2 Cl 2, twice, 1.5 minutes.

6. Forvaskning med 10% EtgN i CHgC^, én gang, 1,5 minutter.6. Pre-wash with 10% EtgN in CH 2 Cl 2, once, 1.5 minutes.

10 7. Neutralisering med 10% Et^N i CHgClg, én gang, 10 minutter.7. Neutralization with 10% Et 2 N in CH 2 Cl 2, once, 10 minutes.

8. Vaskning med CHgClg, tre gange, 1,5 minutter.8. Wash with CHgClg, three times, 1.5 minutes.

9. Tilsætning af BOC-beskyttet aminosyre (5 molært overskud) i dimethylformamid (DMF) og CH2CI2 (1:9 vol/vol).9. Addition of BOC-protected amino acid (5 molar excess) in dimethylformamide (DMF) and CH 2 Cl 2 (1: 9 v / v).

15 10. Tilsætning af DCC i CHgClg (0,5M 5 molært overskud), reaktionstiden var indtil 2 timer.10. Adding DCC in CH 2 Cl 2 (0.5 M 5 molar excess), the reaction time was up to 2 hours.

11. Vaskning med CH2CI2, to gange, 1,5 minutter.11. Wash with CH 2 Cl 2, twice, 1.5 minutes.

Efter koblingsreaktionen testedes en portion af harpiksen med ninhy-20 drin og såfremt der forekom et positivt resultat, blev det antaget, at koblingen var ukomplet og den gentoges med den samme beskyttede aminosyre. Som resultat af en enkelt koblingsreaktion under anvendelse af a-BOC-L-glutamin resulterede følgende peptid-harpiks:Following the coupling reaction, a portion of the ninhydrin resin was tested and, if a positive result occurred, it was assumed that the coupling was incomplete and repeated with the same protected amino acid. As a result of a single coupling reaction using α-BOC-L-glutamine, the following peptide resin resulted:

25 C BZ25 C BZ

of-BOC-GLN-LYS-harpiks hvori BOC er butyloxycarbonyl og CBZ er benzyloxycarbonyl.of-BOC-GLN-LYS resin wherein BOC is butyloxycarbonyl and CBZ is benzyloxycarbonyl.

Denne peptid-harpiks spaltedes og de beskyttende grupper fj'er-30 nedes i et "Kel-F" spaltningsapparat (Peninsula Laboratories, Tnc.) under anvendelse af 10 ml vandfri hydrogenfluorid per gram harpiks ved 0°C i 60 minutter med 5 ml anisol per gram peptid-harpiks som skyllemiddel. Efter inddampning til tørhed i vakuum vaskedes remanensen med vandfri ether. Det rå peptid opløstes i 10% vandig eddi-35 kesyre og filtreredes. Harpiksen vaskedes med 10% vandig eddikesyre og det forenede filtrat opsamledes og lyophiliseredes til dannefse af råt peptid. Det rå peptid rensedes ved modstrøms-fordeling under 149091 16 anvendelse af n-butanol.-eddikesyre:vand (4:1:5) som fordelingsfase til frembringelse af det rene peptid. Det resulterende peptid har følgende sekvens: 05 h-gln-lys-nh2This peptide resin was cleaved and the protecting groups removed in a "Kel-F" cleavage apparatus (Peninsula Laboratories, Tnc.) Using 10 ml of anhydrous hydrogen fluoride per gram of resin at 0 ° C for 60 minutes with 5 ml. anisole per gram of peptide resin as fabric softener. After evaporation to dryness in vacuo, the residue was washed with anhydrous ether. The crude peptide was dissolved in 10% aqueous acetic acid and filtered. The resin was washed with 10% aqueous acetic acid and the combined filtrate collected and lyophilized to form crude peptide. The crude peptide was purified by countercurrent distribution using n-butanol-acetic acid: water (4: 1: 5) as the distribution phase to produce the pure peptide. The resulting peptide has the following sequence: 05 h-gln-lys-nh2

Til identifikation anvendtes tyndtlagskromatografi og elektrofore-se. Aminosyresammensætningen bestemtes ved anvendelse af en amino-syre-analysator.For identification, thin layer chromatography and electrophoresis were used. The amino acid composition was determined using an amino acid analyzer.

10 Tyndtlags kromatografi foretoges på 20 pg prøver pi silikagel (kiselgel, 5 x 20 cm) under anvendelse af 1:1:1:1 n-butanol:eddike-syre:ethylacetat:vand som opløsningsmiddelsystem (R^ ) og på cellulose 6064 (Eastman 20 x 20 cm) under anvendelse af 15:10:3:12 n-buta- 2 1 nol:pyridin:eddikesyre:vand som opløsningsmiddelsystem (R^. ). Rf -15 værdierne for H-ARG-LYS-ASP-VAL-TYR-OH var Rf1 = 1,16 og Rf2 = 0,57. Ninhydrin anvendtes som et spray-reagens.Thin layer chromatography was performed on 20 µg samples in silica gel (silica gel, 5 x 20 cm) using 1: 1: 1: 1 n-butanol: acetic acid: ethyl acetate: water as solvent system (R 2) and on cellulose 6064 ( Eastman 20 x 20 cm) using 15: 10: 3: 12 n-buta-2 1 nol: pyridine: acetic acid: water as a solvent system (R 1). The Rf -15 values for H-ARG-LYS-ASP-VAL-TYR-OH were Rf1 = 1.16 and Rf2 = 0.57. Ninhydrin was used as a spray reagent.

Elektroforese foretoges på en 100 pg prøve på Whitman nr. 3 papir (5,7 x 55 cm) under anvendelse af en pH 5,6 pyridin-acetat-puffer ved en spænding på 1000 V i 1,0 time. Pentapeptidet havde 20 en mobilitet på 2,21 mod katoden for H-ARG-LYS-ASP-VAL-TYR-OH. Ninhydrin- og Pauly-sprayreagenser anvendtes.Electrophoresis was performed on a 100 µg sample on Whitman No. 3 paper (5.7 x 55 cm) using a pH 5.6 pyridine acetate buffer at a voltage of 1000 V for 1.0 hour. The pentapeptide had a mobility of 2.21 toward the cathode of H-ARG-LYS-ASP-VAL-TYR-OH. Ninhydrin and Pauly spray reagents were used.

Eksempel 2Example 2

Til bestemmelse af aktiviteten og egenskaberne af peptidet fra 25 eksempel 1 foretoges bestemmelser på 5-6 uger gamle raske nu/nu mus af begge køn, hvilke mus opfostredes på en BALB/c baggrund (thymocyter udtrykkende Thy-1,2 overfladeantigen) og holdtes under konventionelle betingelser. For antisera, fremstilledes anti Thy-1,2 sera i Thy-1 type mus.To determine the activity and properties of the peptide of Example 1, assays were performed on 5-6 week old healthy now / now mice of both sexes which were raised on a BALB / c background (thymocytes expressing Thy-1,2 surface antigen) and maintained. under conventional conditions. For antisera, anti Thy-1,2 sera were prepared in Thy-1 type mice.

30 Til induktion in vitro af Thy-1+ T-celler eller CR+ B-celle diffe rentiering foretoges induktionen af thymocyt differentiering fra pro-thymocyter in vitro som beskrevet af Komuro og Boyse (Lancet, 1, 740, 1973) under anvendelse af tilvejebringelsen af Thy-1,2 som tegn på T-celle differentiering. Induktionen CR+ B-celle differentiering 35 fra CR~ B-celle prækursorer in vitro foretoges under tilsvarende betingelser under anvendelse som prøvekriterium af evnen af CR+ B-celler til at binde fåre-erythrocyter belagt med subagglutinerende mængder af kanin-antistof og non-lytisk komplement. Milt-celle popu- 149091 17 lationer fra sunde nu/nu mus fraktioneret pi diskontinuerte oksese-rumalbumin-gradienter anvendtes som kilde for begge prækursor-ty-per (Thy-1 og CR ), fordi de har få eller ingen Thy-1+ celler og lavt antal CR+ celler.For in vitro induction of Thy-1 + T cells or CR + B cell differentiation, the induction of thymocyte differentiation from pro-thymocytes in vitro was described as described by Komuro and Boyse (Lancet, 1, 740, 1973) using the provision of Thy-1,2 as evidence of T-cell differentiation. In vitro CR + B cell differentiation from CR ~ B cell precursors in vitro was performed under similar conditions using as a test criterion the ability of CR + B cells to bind sheep erythrocytes coated with subagglutinating amounts of rabbit antibody and non-lytic complement. . Spleen cell populations from healthy now / now mice fractionated in discontinuous oxalicum albumin gradients were used as the source of both precursor types (Thy-1 and CR) because they have few or no Thy-1 + cells and low number of CR + cells.

05 Som resultat af denne bestemmelse blev det fundet, at peptidet udviste en virkningsselektivitet svarende til ubiquitins ved at inducere differentieringen af Thy-1+ T-lymphocyter og af komplement-re-ceptorer (CR ) B-lymphocyter i koncentrationer varierende fra 10 ng/ml til 10 pg/ml.As a result of this assay, the peptide was found to exhibit an activity selectivity similar to ubiquitin by inducing the differentiation of Thy-1 + T lymphocytes and of complement receptors (CR) B lymphocytes at concentrations ranging from 10 ng / ml. ml to 10 pg / ml.

1010

Eksempel 3-7Examples 3-7

Under anvendelse af fremgangsmåden i eksempel 1 og kobling af α-BOC-aminosyrerne i den ønskede rækkefølge efter det i eksempel 1 angivne skema, men med beskyttet sarcosin (fx. or-BOC-sarco- 15 sin) i de passende trin fremstilledes følgende:Using the procedure of Example 1 and coupling the α-BOC amino acids in the desired order according to the scheme set forth in Example 1, but with protected sarcosine (e.g., or-BOC sarcosine) in the appropriate steps, the following were prepared:

Eksempel Forbindelse 3 h-gln-lys-sar-nh2 4 h-sar-gln-lys-nh2 20 5 H-SAR-SAR-GLN-LYS-NH2 6 H-SAR-SAR-SAR-GLN-LYS-NH2 7 H-SAR-SAR-SAR-SAR-GLN-LYS-NH2Example Compound 3 h-gln-lys-sar-nh2 4 h-sar-gln-lys-nh2 5 H-SAR-SAR-GLN-LYS-NH2 6 H-SAR-SAR-SAR-GLN-LYS-NH2 7 H-SAR-SAR-SAR-SAR-GLN-LYS-NH 2

Sekvensen af disse peptider bestemtes med en aminosyre-analy- 25 sator. Tyndtlagskromatografi og elektroforese under samme betingelser som i eksempel 1 gav følgende information: 1 2The sequence of these peptides was determined with an amino acid analyzer. Thin layer chromatography and electrophoresis under the same conditions as in Example 1 gave the following information: 1 2

Eksempel R^ R^ Mobilitet mod katode 3 0/73 0752 1,52 (2 timer) 30 4 1,16 0,57 2,04 5 0,83 0,61 1,88 6 0,5 0,58 1,83 7 0,32 0,54 1,98 (2 timer) 35 Alle R^- og elektroforese-værdier er i forhold til reference-pep- tidet H-ARG-LYS-ASP-VAL-TYR-OH.Example R 2 R 3 Mobility toward cathode 3 0/73 0752 1.52 (2 hours) 30 4 1.16 0.57 2.04 5 0.83 0.61 1.88 6 0.5 0.58 1, 83 7 0.32 0.54 1.98 (2 hours) 35 All R 1 and electrophoresis values are relative to the reference peptide H-ARG-LYS-ASP-VAL-TYR-OH.

18 1A 90 9118 1A 90 91

Eksempel 8Example 8

Under anvendelse af fremgangsmåden i eksempel 2 vurderedes forbindelserne fremstillet ifølge eksempel 3-7 med hensyn til deres evne til at inducere differentieringen af Thy-1+ T-lymphocyter og 05 CR+ B-lymphocyter. Aktivitetsområderne er anført nedenfor:Using the procedure of Example 2, the compounds prepared according to Examples 3-7 were evaluated for their ability to induce the differentiation of Thy-1 + T lymphocytes and 05 CR + B lymphocytes. The activity areas are listed below:

Forbindelse Aktiv koncentrationsområdeCompound Active concentration range

Eksempel nr._ 3 100 pg/ml - 100 ng/ml 10 4 1-10 pg/ml 5 100 pg/ml - 10 ng/ml 6 10 fg/ml - 100 pg/ml 7 1-100 pg/mlExample No. 3 100 pg / ml - 100 ng / ml 10 4 1-10 pg / ml 5 100 pg / ml - 10 ng / ml 6 10 fg / ml - 100 pg / ml 7 1-100 pg / ml

Claims (3)

149091149091 1. Analogifremgangsmåde til fremstilling af peptider indeholdende 05 2 - 7 aminosyrerester, desaminodipeptider eller amider deraf med ubiquitin-lignende aktivitet med den almene formel: A-B 10 hvori A er udvalgt fra gruppen bestående af desamino-GLN, GLN og (SAR)n-GLN, hvor n er et helt tal fra 1 til 4, og B er udvalgt fra gruppen bestående af LYS-OH, LYS-NHg og LYS-SAR-NHg eller farmaceutisk acceptable syreadditionssalte deraf, KENDETEGNET ved, at man 15 i) binder sarcosin eller Ne-beskyttet lysin, der er beskyttet på a-aminogruppen, til en polymer-harpiks ved covalent binding, hvilken polymer er en sådan, som indeholder en funktionel gruppe, hvortil den første beskyttede aminosyre kan bindes fast ved hjælp af den covalente binding,An analogous process for the preparation of peptides containing 05 2 - 7 amino acid residues, desaminodipeptides or amides thereof with ubiquitin-like activity of the general formula: AB 10 wherein A is selected from the group consisting of desamino-GLN, GLN and (SAR) n-GLN wherein n is an integer from 1 to 4 and B is selected from the group consisting of LYS-OH, LYS-NHg and LYS-SAR-NHg, or pharmaceutically acceptable acid addition salts thereof, characterized in that i) binds sarcosine or Non-protected lysine, protected on the α-amino group, to a polymeric resin by covalent bond, which polymer is one containing a functional group to which the first protected amino acid can be bonded by the covalent bond, 20 H) fjerner den α-amino-beskyttende gruppe fra aminosyrehar- piksen og om nødvendigt binder lysin, der er N - og N -beskyttet, til SAR-harpiksen og fjerner α-amino-beskyttelsesgruppen fra LYS-SAR-harpiksen, iii) kobler glutamin eller desaminoglutamin, som er beskyt- 25 tet på dens eventuelle a-aminogruppe og på dens ε-amino- gruppe, til B-aminosyre-harpiksen, og iv) når A-gruppen indeholder en eller flere sarcosyl-dele, fjerner α-aminobeskyttelsesgruppen fra GLN-B-harpiksen, og binder sarcosin, som er beskyttet på dens ot-aminogrup- 3® pe, til kæden og fjerner den α-amino-beskyttende gruppe fra sarcosyl-delen og om nødvendigt gentager denne fremgangsmåde for hver sarcosylrest, der skal kobles, og v) fjerner harpiksen og alle beskyttende grupper fra det resulterende peptid med passende reagenser, og om 33 ønsket fremstiller farmaceutisk acceptable syreadditionssalte af produktet.H) removes the α-amino protecting group from the amino acid resin and, if necessary, binds N- and N-protected lysine to the SAR resin and removes the α-amino protecting group from the LYS-SAR resin; coupling glutamine or desaminoglutamine protected on its optional α-amino group and on its ε-amino group to the B-amino acid resin; and iv) when the A group contains one or more sarcosyl moieties, α the amino protecting group from the GLN-B resin, and binds sarcosine protected on its o-amino group 3 to the chain and removes the α-amino protecting group from the sarcosyl moiety and if necessary repeats this procedure for each sarcosyl residue and v) remove the resin and all protective groups from the resulting peptide with appropriate reagents and, if desired, produce pharmaceutically acceptable acid addition salts of the product.
DK109180A 1979-03-14 1980-03-13 METHOD OF ANALOGUE FOR THE PREPARATION OF PEPTIDS CONTAINING 2-7 AMINO ACID RESIDUES, DESAMINODIPEPTIDES OR AMIDES THEREOF WITH UBIQUITIN SIMILAR ACTIVITY OR PHARMACEUTICAL ACCEPTABLE ACID ADDITIONAL SALTS DK149091C (en)

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US4369137A (en) * 1980-06-17 1983-01-18 Ortho Pharmaceutical Corporation N-Protected pentapeptides useful as intermediates in the preparation of thymopoietin pentapeptide
US4298523A (en) * 1980-06-17 1981-11-03 Ortho Pharmaceutical Corporation Methods and compositions for preparation of H-ARG-X-Z-Y-TYR-R
US4289759A (en) * 1980-06-23 1981-09-15 Ortho Pharmaceutical Corporation Immunoregulatory diketopiperazine compounds
US4481191A (en) * 1983-03-30 1984-11-06 The Regents Of The University Of California Method for controlling blood pressure
WO1986004334A1 (en) * 1985-01-18 1986-07-31 MERCK Patent Gesellschaft mit beschränkter Haftung Immunoregulatory peptides
JPH01104193A (en) * 1986-06-10 1989-04-21 Kyowa Hakko Kogyo Co Ltd Novel polypeptide
US6258550B1 (en) 1993-04-23 2001-07-10 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
WO1994025482A1 (en) * 1993-04-23 1994-11-10 Evans Herbert J Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US5965698A (en) * 1993-04-23 1999-10-12 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
US5952465A (en) * 1993-04-23 1999-09-14 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US5928896A (en) * 1993-04-23 1999-07-27 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein--protein interaction site
US6084066A (en) * 1993-10-29 2000-07-04 Virginia Commonwealth University Polypetides that include conformation-constraining groups which flank a protein-protein interaction site
US5770720A (en) * 1995-08-30 1998-06-23 Barnes-Jewish Hospital Ubiquitin conjugating enzymes having transcriptional repressor activity

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US4113858A (en) * 1975-01-20 1978-09-12 St. Luke's Hospital Novel compounds, compositions and methods of their use
FR2391994A1 (en) * 1977-05-25 1978-12-22 Anvar Polypeptide analogues of serum thymus factor - with thymus hormonal or antagonistic activity
SE446866B (en) * 1977-11-15 1986-10-13 Sloan Kettering Inst Cancer SET TO MAKE AN ACTIVE POLYPEPTIDE WITH THE ABILITY TO INDUCE DIFFERENTIZATION OF BAD T-Lymphocytes AND COMPLEMENT RECEPTOR (CR? 72+) B-Lymphocytes
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FI800779A (en) 1980-09-15
ES8103026A1 (en) 1981-02-16
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NO149998C (en) 1984-08-01
IE800515L (en) 1980-09-14
EP0016612B1 (en) 1982-11-24
DK109180A (en) 1980-09-15
IE49423B1 (en) 1985-10-02
YU41913B (en) 1988-02-29
GR68038B (en) 1981-10-27
FI67691C (en) 1985-05-10
JPH0153268B2 (en) 1989-11-13
CA1146166A (en) 1983-05-10
ES489521A0 (en) 1981-02-16
PT70948A (en) 1980-04-01
IL59603A (en) 1983-02-23
YU65980A (en) 1984-02-29
FI67691B (en) 1985-01-31
ATE1856T1 (en) 1982-12-15
NO800724L (en) 1980-09-15
DE3061127D1 (en) 1982-12-30
IL59603A0 (en) 1980-06-30
JPS55143946A (en) 1980-11-10
PT70948B (en) 1981-06-24
EP0016612A2 (en) 1980-10-01

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